Magnetic compatible microsphere for purifying thrombin and preparation method and application thereof

A thrombin and affinity technology, applied in chemical instruments and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of high quality requirements of sample loading liquid, time-consuming separation of thrombin, and low chromatographic efficiency, etc. Achieve the effect of saving production investment, fewer steps, saving time and cost

Active Publication Date: 2010-06-02
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, one of the limitations of affinity column chromatography is the high production cost and the high quality requirements of the sample liquid.
During biochemical extraction, the sample liquid obtained by rough separation in the early stage often contains suspended particles and various impurities. If it is not pre-purified, problems such as clogging and low chromatographic efficiency will occur and affect the quality of the product.
In addition, the separation of thrombin by affinity chromatography is time-consuming and produces a large volume of dilute protein solution, which has a great impact on production and preparation
At present, there is no report on the rapid purification of thrombin using affinity magnetic separation technology at home and abroad.

Method used

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  • Magnetic compatible microsphere for purifying thrombin and preparation method and application thereof
  • Magnetic compatible microsphere for purifying thrombin and preparation method and application thereof
  • Magnetic compatible microsphere for purifying thrombin and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Preparation of magnetic affinity microspheres for purified thrombin

[0040] 1) Fe 3 o 4 Preparation of nano-magnetic cores: Measure FeCl separately 2 and FeCl 3 Mix the solution evenly, then add PEG4000 (polyethylene glycol 4000, product number: Topbio, Merck817006), fully dissolve and quickly add it dropwise to 3mol / l NaOH under magnetic stirring, the solution quickly becomes thick and bright black, showed that the hydrolysis produced a large amount of Fe 3 o 4 , maintain the pH value of the solution at 9-11, continue to stir for 30 minutes to make the large particles smaller, then sonicate for 10 minutes to dissolve the small particles, mature in a water bath at 80°C for 30 minutes, and then sonicate for 10 minutes. Use a magnet to hold the bottom and pour off the supernatant, rinse with double-distilled water until neutral, filter with suction, and vacuum freeze-dry for later use.

[0041] 2) Chitosan-wrapped nano-magnetic core: accurately weigh 1.0...

Embodiment 2

[0043] Example 2. Separation and Purification of Thrombin Using Magnetic Affinity Microspheres for Purification of Thrombin

[0044] 1) Separation and purification of thrombin by chitosan magnetic affinity microspheres: the prepared chitosan magnetic affinity microspheres are swelled by adding appropriate amount of distilled water, and fully equilibrated with 0.05mol / L, pH7.5 Tris-HCl buffer After washing, add thrombin crude enzyme solution (preliminary purification of fresh anticoagulated plasma by barium citrate method), slowly stir and adsorb at room temperature for 30 minutes, and separate the adsorption solution with a magnet. Wash the microspheres with the above-mentioned buffer solution containing 0.1mol / L NaCl to remove impurities, then use the Tris-HCl buffer solution containing 1mol / L NaCl as the eluent to wash the microspheres, collect the eluate through magnetic separation, and dialyze After desalination, concentration, and vacuum freeze-drying, the final product w...

Embodiment 3

[0054] Example 3. Detection of construction and performance of magnetic affinity microspheres

[0055] The present invention will be further described below in conjunction with characterization technique method:

[0056] 1. Powder X-ray Diffraction (XRD) analysis

[0057] The prepared Fe 3 o 4 Nano magnetic nuclei and magnetic affinity microspheres are analyzed, using Cu (Ka, λ=0.15406nm) target, graphite monochromator (Fourier transform infrared spectroscopy FTS 3000 American DIGILAB), working voltage 40kv, working current 60mA, scanning speed 80 / min.

[0058] Depend on figure 1 It can be seen that the prepared Fe 3 o 4 The XRD diffraction peaks of nanoparticles (a) and magnetic affinity microspheres (b) have six characteristic peaks, at 2θ=30.06, 35.44, 43.10, 53.74, 57.10, 62.74, corresponding to Fe 3 0 4 (220), (311), (400), (422), (511) and (440) crystal faces, which are consistent with the standard card control (JCPDS card no.79-0418) of ferric oxide, indicating ...

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Abstract

The invention provides a magnetic compatible microsphere for purifying thrombin and a preparation method and application thereof. The magnetic compatible microsphere for purifying the thrombin comprises a magnetic core, a polymer enwrapping material enwrapped outside the magnetic core, and a ligand coupled with the polymer enwrapping material. The magnetic affinity microsphere is characterized in that the ligand can be specifically compatible with the thrombin.

Description

technical field [0001] The present invention relates to the purification of proteins, in particular, the purification of thrombin. Background technique [0002] Thrombin (EC3.4.21.5) is a highly specific serine proteolytic enzyme that can hydrolyze the four Arg-Gly peptide bonds of fibrinogen to produce insoluble fibrin, making the blood become clotted In clinical practice, thrombin is the drug of choice for local hemostasis, and can be used for hemostasis of small blood vessels that are difficult to ligate during surgery, gastrointestinal bleeding, and traumatic bleeding. During implementation, it is often dissolved in dry powder or normal saline and applied locally to wounds and surgical sites, which can quickly achieve the purpose of hemostasis. For gingival and nasal bleeding caused by abnormal coagulation factors, when other drugs fail to achieve the hemostatic effect, thrombin can be used to directly act on the bleeding site. Current research also shows that thrombin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/28B01J20/285B01J20/30C12N9/74
Inventor 董先智李梅基李昭华
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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